PROJECT SUMMARY Glaucoma is the most common cause of irreversible blindness and will affect more than 100 million people between 40 to 80 years old by 2040. It causes severe visual loss due to degeneration of optic nerve (ON) and retinal ganglion cells (RGCs). There is a significant unmet clinical need for neuroprotectants. Our previous studies of ON traumatic injury and glaucoma demonstrated that both acute and chronic ON injury induce endoplasmic reticulum (ER) stress in RGCs. We were able to protect the injured RGC soma and axons if we blocked the detrimental effects of ER stress by manipulating two key downstream molecules of the unfolded protein response (UPR) in opposite ways: a) deletion of CCAAT/enhancer binding protein homologous protein (CHOP), and/or b) activation of X-box binding protein 1 (XBP-1). Thus axon injury-induced ER stress may be a common mechanism of neuronal damage and targeting neuronal ER stress may have considerable therapeutic neuroprotective potential in diseases associated with axonopathy. As the first step, we propose to identify novel ER stress modulators by screening chemical libraries with cell-based high throughput screen (HTS) assays; and then to validate whether these agents promote RGC and ON survival and preserve visual function in mouse glaucoma models. Recently, exciting recent studies of axonal Wallerian degeneration have shown that several key molecules involved in axonal NAD+ metabolism are critical for axonal degeneration. SARM1 (Sterile Alpha and TIR Motif 1), for example, is negatively regulated by axonal NAD+ synthetic enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) to induce axon degeneration; deletion of SARM1 or activation of axonal NMNATs results in axon protection. Thus, we will test the hypothesis that modulating both intrinsic neuronal ER stress and NAD+ metabolism will synergistically prevent both RGC soma and axon (ON) degeneration and preserve vision in glaucoma. This study may generate novel combinatory therapeutic strategies that lead to more efficient neuroprotection in patients. And finally, we will develop novel in vivo imaging tools for RGC morphology and function studies and acquire much needed insights into the mechanism of RGC ER stress initiation. We expect the results through these studies will provide essential information for clinical application of ER stress modulation, and establish translatable techniques and biomarkers that will greatly facilitate clinical management of glaucoma patients.